Research paper
Identification of a candidate therapeutic antibody for treatment of canine B-cell lymphoma

https://doi.org/10.1016/j.vetimm.2015.02.004Get rights and content

Abstract

B-cell lymphoma is one of the most frequently observed non-cutaneous neoplasms in dogs. For both human and canine BCL, the standard of care treatment typically involves a combination chemotherapy, e.g. “CHOP” therapy. Treatment for human lymphoma greatly benefited from the addition of anti-CD20 targeted biological therapeutics to these chemotherapy protocols; this type of therapeutic has not been available to the veterinary oncologist. Here, we describe the generation and characterization of a rituximab-like anti-CD20 antibody intended as a candidate treatment for canine B-cell lymphoma. A panel of anti-canine CD20 monoclonal antibodies was generated using a mouse hybridoma approach. Mouse monoclonal antibody 1E4 was selected for construction of a canine chimeric molecule based on its rank ordering in a flow cytometry-based affinity assay. 1E4 binds to approximately the same location in the extracellular domain of CD20 as rituximab, and 1E4-based chimeric antibodies co-stain canine B cells in flow cytometric analysis of canine leukocytes using an anti-canine CD21 antibody. We show that two of the four reported canine IgG subclasses (cIgGB and cIgGC) can bind to canine CD16a, a receptor involved in antibody-dependent cellular cytotoxicity (ADCC). Chimeric monoclonal antibodies were assembled using canine heavy chain constant regions that incorporated the appropriate effector function along with the mouse monoclonal 1E4 anti-canine CD20 variable regions, and expressed in CHO cells. We observed that 1E4-cIgGB and 1E4-cIgGC significantly deplete B-cell levels in healthy beagle dogs. The in vivo half-life of 1E4-cIgGB in a healthy dog was ∼14 days. The antibody 1E4-cIgGB has been selected for further testing and development as an agent for the treatment of canine B-cell lymphoma.

Introduction

Canine lymphoma is a significant veterinary problem, comprising up to a quarter of all canine cancers. The majority of canine lymphomas (≥60%) are of B-cell origin (B-cell lymphoma; BCL), while the remainder are T-cell derived or of mixed B-and T-cell immunophenotype (Modiano et al., 2005). The current standard of care for canine BCL is a protocol employing several chemotherapeutic drugs, commonly cyclophosphamide, hydroxydaunorubicin/doxorubicin, oncovin/vincristine and prednisone (CHOP therapy; Garrett et al., 2008). Left untreated, dogs with lymphoma typically only survive about 6 weeks from time of diagnosis. CHOP therapy extends the survival time of dogs with BCL to 10–14 months (Richards and Suter, 2015). Most dogs experience complete remission followed by one or more relapses. CHOP therapy is rarely curative in dogs, possibly because veterinary protocols tend to be less dose-intensive as compared to human CHOP protocols; in fact, only ∼20% of treated dogs will survive beyond 2 years (Richards and Suter, 2015).

The most common form of BCL in humans is non-Hodgkins lymphoma (NHL). The advent of the monoclonal antibody rituximab revolutionized human BCL treatment by significantly increasing response and survival rates in NHL patients over chemotherapy alone. The standard of care for human NHL is now rituximab in combination with CHOP (R-CHOP) (Dotan et al., 2010, Molina, 2008). The target of rituximab is CD20, a tetra-membrane spanning protein expressed on B-cell lymphomas and normal B-cells from the pre-B-cell through memory B-cell stages. The fact that CD20 is not expressed on plasma cells makes it a particularly attractive therapeutic target, as depletion of CD20-positive B-cells does not eliminate the antibody response to foreign pathogens (van Meerten and Hagenbeek, 2010). The actual function of CD20 is not well understood, but it may play a role in Ca2+ transport across the plasma membrane (Bubien et al., 1993, Li et al., 2003).

Rituximab is a chimeric antibody, composed of mouse variable regions that bind to CD20 and a human IgG1 constant/Fc region that mediates effector functions of the antibody. Rituximab works by depleting CD20-positive B-cells, largely via antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) (Beers et al., 2010). It has been shown that, as is the case in humans, canine B-cell lymphomas express CD20 but canine T cell lymphomas do not (Jubala et al., 2005, Kano et al., 2005). For this reason, a rituximab-like therapeutic specific to canine CD20 could potentially be effective in treating BCL in dogs. Rituximab does not bind to canine CD20, likely due to a lack of conservation of the rituximab epitope in the canine protein (Impellizeri et al., 2006, Jubala et al., 2005), making it impossible to use rituximab for treatment of canine BCL. Here, we describe the generation and characterization of a chimeric anti-CD20 antibody specifically designed for therapeutic B-cell depletion in dogs. This reagent should enable the evaluation of the effectiveness of anti-CD20 therapy for BCL in dogs.

Section snippets

Cloning and expression of canine and human CD20 ECD2 fusion proteins and hybrids

The second extracellular domain (ECD2) of canine and human CD20 was cloned by RT-PCR. RNA was isolated from canine or human PBMCs (Bioreclamation, Westbury, NY) using Qiagen RNeasy and QIAshredder kits (Qiagen, Valencia, CA). The ThermoScript RT-PCR system (Life Technologies, Carlsbad, CA) was used to generate cDNA using random hexamers. Canine CD20 was amplified from cDNA using primers BE-001 and BE-003, and human CD20 was amplified from human cDNA using primers BE-142 and BE-147. PCR products

Generation of monoclonal antibodies against canine CD20

A hybridoma approach was used to make monoclonal antibodies against canine CD20. The majority of human therapeutic anti-CD20 antibodies (including rituximab) bind to the larger second extracellular domain (ECD2) of CD20 (Beers et al., 2010). For this reason, ECD2 of canine CD20 was cloned from canine PBMCs and inserted into a mouse Fc fusion vector. Purified protein produced from the resulting construct, referred to as canine CD20 ECD2-mFc, was used to immunize mice in a RIMMS protocol.

Discussion

Knowledge about differences in effector function between human IgG subclasses has proven invaluable in the design of human biologics, enabling both fit-for-purpose selection of human Fc's and Fc engineering. To our knowledge, this is the first report comparing the in vivo behavior of different canine isotypes. While the exact mechanism(s) of B-cell depletion by the 1E4-cIgGB and 1E4-cIgGC anti-CD20 antibodies we have developed in this study are under investigation, our B-cell depletion and

Acknowledgements

We thank Chris Trussell, Fatou Sow, and Michelle Gaylord for assistance with flow cytometry, Robert Dunn for secondary antibody recommendations, Brenda Philips for supplying tumor tissue, and Thomas Jostock, Zorica Dragic, and Lori Jennings for guidance with stable pool generation.

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    1

    These authors contributed equally to this paper.

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    Current address: Inhibrx, 11099 North Torrey Pines Road, Suite 280, La Jolla, CA 92037, United States.

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    Current address: COI Pharmaceutical, 11099 North Torrey Pines Road, Suite 290, La Jolla, CA 92037, United States.

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